In this study, we have performed a comprehensive quantitative proteomic study to analyze aortic proteins from young (8 mo) and old (30 mo) rats. Using 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE), we have obtained 2-D gel maps of 301 identified non-redundant proteins from rat aorta and observed 18 proteins that significantly change abundance with aging. Utilizing Isobaric tags for relative and absolute quantitation (iTRAQ), 921 proteins were quantified and between both methods, 50 proteins were shown to have significantly different age-associated abundance. Proteomic analysis shows that one protein of interest, MFG-E8, significantly increases in abundance in old rat aortae. Transcription and translation analysis demonstrated that aortic MFG-E8 mRNA and protein levels increase with aging in several mammalian species, including humans. Dual immunolabeling shows that MFG-E8 colocalizes with both angiotensin II (Ang II) and monocyte chemoattractant protein-1 (MCP-1) within vascular smooth muscle cells (VSMC) of the thickened aged aortic wall. Exposure of early passage VMSC from young aorta to Ang II markedly increases MFG-E8 and enhances invasive capacity to levels observed in VSMC from old rats. Treatment of VSMC with MFG-E8 increases MCP-1 and VSMC invasion that are inhibited by the MCP-1 receptor blocker, vCCI. Silencing MFG-E8 RNA substantially reduces MFG-E8 expression and VSMC invasion capacity. Thus, arterial MFG-E8 significantly increases with aging and is a pivotal relay element within the Ang II MCP-1/VSMC invasion signaling cascade. We have identified that aging arterial MFG-E8-enriched VSMC are activated and proliferating both in vivo and in vitro. Increased MFG-E8 in VSMC triggers phosphorylation of ERK-1/2, augments levels of PCNA and CDK4, increases BrdU incorporation and promotes growth. The knockdown of MFG-E8 reduces the rate of cell cycling, accelerating signaling molecules PCNA and CDK4 expression, facilitating cell entry into a growth-arrested state. Furthermore, we find that &#945;v&#946;5 and PDGF receptor are upregulated with MFG-E8 and also are elements to relay proliferative signals to aging VSMC. In addition, MFG-E8 facilitates the profibrosis of VSMC with aging via an interaction with caveolin 1 (CAV1) signaling. Exposure of isolated aortic VSMC of old rats (30-mo) to MFG-E8 increases the profibrogenic signaling molecules TGF-&#946;1, TGF-&#946; receptor type II (T&#946;RII), p-SMAD-2/3, and collagen I (Col I) while knockdown of MFG-E8 gene expression substantially reduces the expression of these molecules. Both co-immuno-labeling and co-immune-precipitation of MFG-E8, T&#946;RII, and CAV1 in young VSMC (8-mo) indicates their physical interactions. In young VSMC, MFG-E8 up-regulates expression of CAV1. Knockdown of the CAV1 gene, similar to MFG-E8 exposure, significantly increases the expression of TGF-&#946;1, T&#946;RII, p-SMAD-2/3, and Col I. Over-expression of the CAV1 gene, similar to MFG-E8 gene knockdown, markedly decreases the expression of these fibrogenic molecules in both young and old VSMC. Furthermore, MFG-E8 treatment significantly increases the expression of TGF-&#946;1, p-SMAD-2/3, and Col I in CAV1 silenced young VSMC. Interestingly, MFG-E8 exposure does not increase expression of these profibrogenic molecules in old VSMCs when CAV1 is overexpressed. These results, for the first time, demonstrate that MFG-E8 modulates TGF-&#946;1 fibrogenic signaling in VSMC in a CAV1/age-dependent manner. Importantly, we find that the involvement of MFG-E8 in atherosclerotic lesions increases with age. In in vivo studies: 1) Immunostaining demonstrates that MFG-E8 protein in the aortic wall in ApoE-/- mice on a standard chow diet increases with age and is localized predominantly in advanced plaques and the elastin laminae . 2) In nonhuman primates (9 to 23- years old), iTRAQ assay indicates that levels of arterial MFG-E8 protein are highly correlated with age. Arterial MFG-E8 protein expression in older monkeys (>17-years-old) fed a high cholesterol diet for 2 years is further increased 1.6-fold compared to older animals on a standard diet. 3) In aortic thoracic intimae isolated from adult humans (age 22-to 69-years old), Western blot analysis indicates that MFG-E8 protein abundance is significantly increased in atherosclerotic areas compared to the adjoining grossly normal intimae. Notably, the glycosylated form of MFG-E8 is markedly increased in lesion areas and rarely detected in normal intimae. 4) Immunostaining indicates that MFG-E8 is mainly derived from smooth muscle cells (alpha-smooth actin positive) and macrophages (CD 68 positive). In in vitro studies: 1) Early passage thoracic aortic smooth muscle cells (SMC) isolated from young and old monkeys, and exposed to pathogenic oxidative low density lipoprotein (oxLDL) shows that the uptake capacity of lipid in old cells exceeds that of young cells, facilitating trans-differentiation into foam-like cells (CD68 positive). 2) Silencing of MFG-E8 RNA markedly reduced the uptake of oxLDL into SMC. In addition, an MFG-E8 fragment, medin, plays an important role in arterial aging and athersocelrosis. The effects of medin on the behavior of VSMCs with advancing age are under investigation. Taken together, MFG-E8 translation and glycosylation and its fragment medin are increased in atherosclerotic aortic walls with aging in various species, including humans, potentially playing a precipitating role in the formation of foam cells, the hallmark of atherosclerosis.